Production Of Immunogenic Virus Structural Proteins By Goat Mammary Gland Bioreactor

Contagious diseases of animals can present significant harm to the development of the modern livestock industry and cause significant economic loses worldwide. Furthermore, some zoonoses caused disease and death in people. Vaccination continues to be the most costeffective approach to prevent these diseases. With the development of molecular biotechnology, recombinant protective antigens of pathogen purifying from different bioreactors have served as subunit vaccines with more biological stability, cost effectiveness and safety, which may offer a means to significantly improve the veterinary vaccines. Mammary gland bioreactor is an ideal platform for producing exogenous proteins, because it can produce complex, biologically active proteins in an efficient and economic manner. This study aims to develop an efficient approach for production of immunogenic virus structural proteins by goat mammary gland bioreactor,which may lay the foundation for production of subunit vaccines. Using TALE nickase-mediated gene targeting, exogenous genes encoding 5 different structural proteins of CSFV, FMDV and PRRSV were site-specifically integrated into the exon 2 of goat β-lactoglobulin(BLG) gene and gene-targeted goats were produced by somatic nuclear transfer(SCNT). Immunization test in mice indicated that recombinant proteins purifying from milk of transgenic animals exhibit the satisfied immunogenicity, and can excite humoral immunity. The main contents of this research are as follows:1. Design and construction of TALENs targeting the exon 2 of goat BLG geneThe target site for TALENs against exon 2 of the goat BLG gene was designed by “TAL Effector-Nucleotide Targeter(TALE-NT) 2.0”, and the DNA constructs of TALENs, pTALENE2-L and pTALEN-E2-R were produced by the “unit assembly” approach. An RFP-GFP reporter system was used to determine the nuclease activities of pTSBE1-R and pTRBE1-L in 293 FT cells. The cells transfected with TALENs and their corresponding reporter construct yielded appreciable levels of red and green fluorescence, which indicated that these designed nucleases specifically cleaved the target site. The dual fluorescent reporter vectors containing the target site and its homologous sequence of bovine BLG gene were constructed and cotransfected with TALENs into HEK293. The fluorescent expression indicated that theses TALENs against BLG exon 2 could specifically cleaved the target site. TALE nickase were generated by introducing a mutation at the amino acid D450(Asp) of FokⅠin pTALEN-E2-L which resulted in Asp turning to Ala.2. Comparation of EGFP expression in two different integration sites of BLG locusGene targeting vectors were designed according to the targeting sites of TALENs against BLG exon 1 and TALENs against BLG exon 2. The homologous DNA fragments, EGFP fragment and bGHpA fragment were PCR amplified and used to construct targeting vectors based on ploxpⅡ. The targeting vectors, pBTE1-EGFP and pBTE2-EGFP were against BLG exon 1 and BLG exon 2, respectively. TALENs and their corresponding EGFP targeting vectors were co-transfected to goat mammary epithelial cells(GMEC). 12 and 14 cell clones with EGFP site-specifically integrated into BLG exon 1 and 2 were obtained by G418 selection and PCR analysis, respectively. Real-time quantification PCR and Western analysis of the targeted GMECs induced by hormone in vitro demonstrated that EGFP could be expressed in all of the cells with EGFP gene site-specific integration, and the cells with EGFP gene integrated into BLG exon 2 exhibited the better ability for EGFP expression. The results indicated that exogenous genes site-specifically integrated into BLG locus can be expressed in GMECs and external secreted under control of the endogenous BLG regulatory region. Furthermore, the intron 1 of BLG gene enhanced the expression of exogenous gene.3. Generation of transgenic goats with viral subunit proteins genes targeted in BLG exon 2 by TALE nickase and SCNTThe coding sequence of PRRSV GP5-M, CSFV E0, CSFV E2, A-FMDV VP1 and O-FMDV VP1 were amplified by PCR, and the bGHpA was respectively fused to the 3’ terminal of these fragments to construct gene targeting vectors pBTE2- GP5-M、pBTE2-E0、pBTE2-E2、pBTE2-VP1_A and pBTE2-VP1_O. The mRNAs encoding TALE Nickase were transcribed in vitro and co-transfected with targeting vectors into goat fetal fibroblasts. After selection with G418, the cell clones with site-specific integration of BLG exon 2 were obtained by PCR analysis and selection with GANC. Using the targeted cell clones as nuclear donor, 7 transgenic goats were produced by SCNT. Genotype analysis by PCR, DNA sequencing and Southern blot showed that there were 2 GP5-M gene targeted goats,2 E0 gene targeted goats, 1 E2 gene targeted goat and 2 VP1_A gene targeted goats. These results demonstrated that TALE nickase-mediated gene targeting can be used to introduce site-specifically integration of exogenous genes in goat somatic cells.4. Evaluation of recombinant proteins in targeted goats milk and verification of their immunogenicityA-FMDV VP1 and CSFV E2 gene targeted goats were induced to lactation by hormone injection. Serum milk was generated by taking off fat and casein from the transgenic milk. The expression of recombinant VP1(rVP1) and recombinant E2(rE2) was confermed by Coomassie brilliant blue, Western blot and ELISA analysis, and the concentrations of rVP1 and rE2 were 1.02 mg/mL and 1.46 mg/mL, respectively. rVP1 and rE2 were purified from serum milk by Ni-ion affinity chromatography, and injected with adjuvant to Balb/c mice for immunogenicity detection. At four weeks after vaccination, the FMDV-antibody and CSFV-antibody could be detected in serum of immuned mice with rVP1 and rE2, respectively. These results indicated that the exogenous genes site-specifically integrated into goat BLG exon2 can be expressed in mammary gland and secreted into milk under control of the endogenous BLG regulatory region. In addition, the recombinant structural proteins exhibited satisfied immunogenicity to excite the humoral immune response.In conclusion, transgenic goats were generated by TALE nickcase-mediated gene targeting and SCNT, two of the transgenic animals were induced lactation by injecting hormones, the purified recombinant structural proteins of virus from the milk were confirmed with the immunogenicity, which will be helpful for research of subunit veterinary vaccines.